The present invention relates to novel derivatives of chlorophyll (Chl) and of bacteriochlorophyll (Bchl), to their preparation and their application in photodynamic therapy, as well as in fluorescence detection and monitoring techniques.
During the last two decades, there has been an increasing interest in the utilization of photosensitizers for cancer therapy. In this technique, known as photodynamic therapy (PDT), singlet oxygen, oxygen radicals and superoxides or peroxides are produced by in situ photosensitization of previously applied chromophores and intoxicate the malignant cells (Dougherty, 1987). The technique utilizes non-toxic drugs in combination with non-hazardous photosensitizing irradiation, and has the potential of being more selective yet no less destructive when compared with the commonly used chemotherapy or radiotherapy, and therefore it is expected to increase the quality of life of the treated patients.
The photosensitizers used in PDT need to have a high quantum yield for singlet oxygen production and high affinity and selectivity to the malignant tissue. Porphyrins have a relatively high quantum yield for the formation of an excited triplet state and the difference between the energies of this state and their singlet ground state makes them good energy donors to excite the ground state (triplet) oxygen to its singlet state. It has been known for some time that hematoporphyrin (HP) (FIG. 1) and hematoporphyrin derivative (HPD) tend to accumulate in neoplastic tissues. Thus, HP and HPD mixtures have become the preferred compounds for PDT.
The commercially used HPD mixture as photodynamic agent, Photofrin II (Quarda Logic Technologies, Inc., Vancouver, BC, Canada) contains a high proportion of ether-linked HP oligomers that have high extinction coefficient values around 400 nm (the so-called Soret band), but much smaller values in the visible (500-630 nm; the so-called Q bands). Unfortunately, due to the extensive attenuation of UV-Visible light by the animal tissue, the quantum yield of photosensitization by HPD in situ is very low. Therefore, intensive illumination and large amounts of HPD are required for efficient treatment of tumors. As the amount of applied HPD increases, the chances of its accumulation in normal tissues and the accompanying risk of damaging non-malignant sites, profoundly increases. An additional disadvantage of HPD related analogues is their slow clearance from the human body. Patients treated with HPD suffer from skin phototoxicity over periods of weeks.
The strong attenuation of UV-VIS light by the animal tissue and the limited specificity of HPD to the malignant sites have motivated research and synthesis of new phototherapeutic agents that absorb light beyond 650 nm and have increased retention in the malignant site (McRobert et al., 1989).
In order to increase the retention in, and the specificity to, malignant tissues, various porphyrin derivatives containing particular chemical groups attached to the pyrrole residues of the porphyrin structure have been tested. The red shift of the compounds absorption relative to HPD is achieved by variations in the porphyrin xcfx80-electron system.
Following this approach, there has been an increasing interest in using Chl and Bchl derivatives as PDT agents (Kreimer-Birnbaum, 1989; Spike and Bommer, 1991). Chls and Bchls are di- and tetrahydroporphyrin derivatives, respectively, consisting of 4 pyrrole and one isocyclic rings linked to each other and to the atom of Mg, as depicted in FIG. 2 for chlorophyll a (Chla) and in FIG. 3 for bacteriochlorophyll a (Bchla), M representing Mg and the radical R being phytyl or geranylgeranyl in Bchla, and phytyl in Chla. The Bchla molecule differs from the Chla molecule by having two more xcex2-carbon (peripheral carbons of the pyrrole rings) reduced. The variety of Chls and Bchls results from the variation of substituents at the macrocycle or the alcohol residue that esterifies the 17-propionic acid residue. In the naturally occurring Bchla, the alcohol residue is phytyl or geranylgeranyl, while in Bchlb, for example, it is geranylgeranyl. The acids derived from chlorophyll and bacteriochlorophyll are designated chlorophyllide (Chlide) and bacteriochlorophyllide (Bchlide), respectively. The free acids derived from Chla and Bchla are designated Chlidea and Bchlidea, respectively. The compounds derived from Chl and Bchl devoid of a central metal atom are designated pheophytin (Pheo) and bacteriopheophytin (Bpheo), respectively. The pheophytins derived from Chla and Bchla are designated Pheoa and Bpheoa, respectively. The free acids derived from pheophytin and bacteriopheophytin are designated pheophorbide and bacteriopheophorbide, respectively.
The Chls and the Bchls harvest solar energy and initiate electron transfer in biological photosynthesis. Their lowest-energy transitions (the so-called Qy transitions) in the monomeric forms are found at 670-800 nm and can be shifted up to 100 nm in aggregated forms. These transitions have extremely high extinction coefficients. The probability of inter-system crossing from the excited singlet state of the Chls and Bchls to their lowest triplet state is fairly high (30-50%) and assures a high yield of excited oxygen molecules. In fact, the photosensitization of oxygen by Chls and Bchls is underlined by the fact that they are involved in important degradative processes in photosynthetic bacteria and plants and therefore all photosynthetic organisms have a variety of protective mechanisms against singlet oxygen or oxygen radicals. Since Chls and Bchls are natural compounds that are ordinarily consumed by animals, their in vivo degradation is very fast relative to HP or HPD (Llewellyn, et al. 1990). This is an important advantage that reduces the patient subjection to prolonged irradiation.
Yet, there are several problems in using the native Chl or Bchl extracts for PDT. First, they are hard to deliver to the malignant site because they are strongly hydrophobic. Second, they are very labile under normal delivery conditions, i.e., in the presence of oxygen at room temperature and under normal light conditions. Third, they have no moiety to target them specifically to the malignant tissue. Due to these limitations, the potential of these photosynthetic pigments as sensitizers in PDT is presently hard to realize. It would be highly desirable to synthesize Chl and Bchl derivatives that would overcome these difficulties and could be successfully used in PDT. It is the object of the present invention to provide such derivatives.
The side groups of Chl and Bchl determine the molecule""s overall affinity to different environments.
It has now been found according to the present invention that new Chl and Bchl derivatives can be prepared for use as photosensitizers in therapy and diagnostics by modification of the ester group at the C17-propionic acid side group of the Chl or Bchl structure.
The present invention thus relates to new Chl and Bchl derivatives of the general formula I
Xxe2x80x94COxe2x80x94Yxe2x80x94Axe2x80x94Rxe2x80x83xe2x80x83I
in which
Xxe2x80x94COxe2x80x94 is a C17-propionyl residue of a compound selected from the group consisting of a chlorophyll (Chl), a bacteriochlorophyll (Bchl), and a Chl and a Bchl in which the central Mg atom is substituted by a different divalent metal atom;
Y is O, S or NH;
A is a covalent bond or a straight or branched, saturated or unsaturated, substituted by one or more functional groups selected from the group consisting of OH, COOH, NH2, and CONH2, or unsubstituted hydrocarbon chain having from 2 to 20 carbon atoms and containing an end functional group selected from the group consisting of OH, COOH and NH2; or such a hydrocarbon chain interrupted by one or more heteroatoms selected from the group consisting of O, S or N, or by a phenyl ring; and
R is a residue of a peptide or a protein that are cell-specific ligands.
The term xe2x80x9cChl or Bchl residuexe2x80x9d herein means any derivative of Chl or Bchl, both natural and synthetic derivatives, and includes the compound in which the central Mg atom has been substituted by other divalent metals, such as Zn, V, Cu, Co, Ni or Sn. Bchl derivatives are preferred according to the invention.
In a preferred embodiment, Y is O and Xxe2x80x94COxe2x80x94 is the residue of Chla or Bchla, shown in FIGS. 2 and 3, respectively.
The hydrocarbon chain A has preferably from 5 to 20 carbon atoms. It may be saturated or unsaturated, interrupted by heteroatoms, such as O, N and/or S or by phenyl, and/or substituted by functional groups consisting of OH, NH2, CONH2 and COOH.
The radical R may be derived from an amino acid or a derivative thereof, such as serine, tyrosine and lysine and derivatives thereof, e.g., L-serine or tyrosine methyl esters, or from a peptide, e.g., seryl serine methyl ester, melanocyte stimulating hormones (MSH), or from a protein.
In particular it is envisaged by the invention the conjugation of Chl and Bchl with different amino acids, and further conjugation of these Chl/Bchl amino acid conjugates with hormones, growth factors or derivatives thereof, or tumor-specific antibodies, or any other cell specific ligands, thus obtaining suitable site-directed photosensitizers (SPD).
Examples of peptide/protein hormones for use in the invention is one of the melanocyte-stimulating hormones (MSH) (melanotropins), such as xcex1-, xcex2- or xcex3-MSH, that specifically bind to receptors in the melanocytes, and can thus target the photosensitizing moiety into melanoma tumors, and neurotensin, somatostatin, human chronic gonadothropin (hCG), epidermal growth factor and immunoglobulin-G.
The invention further comprises processes for the preparation of the compounds of formula (I).
Also included in the invention are compositions comprising a compound of formula (I) for diagnostic and photodynamic therapy purposes, and a method of photodynamic therapy of cancer patients which comprises treatment of the patient with a compound of formula (I) followed by local irradiation.
A method according to the invention for locating a tumor by localization of a suspected area comprises: (a) administering to a subject suspected of having a tumor a compound of formula I; and (b) measuring the fluorescence of the suspected area after 4-6 hours by standard procedures, wherein a higher fluorescence indicates tumor sites. Another method according to the invention for tumor diagnosis comprises: (a) administering to a subject suspected of having a tumor a compound of formula I; and (b) imaging the subject by standard procedures, wherein accumulation of the radioactive material indicates tumor sites.
A method of photodynamic therapy of tumors and metastatic tumors according to the invention consists of injecting into a patient an appropriate amount of a compound of formula I, followed by local irradiation.
For the treatment of melanoma tumors the patient is injected with an appropriate amount of a melanocyte-stimulating hormone, followed by local irradiation.
The invention further includes a method of photodynamic therapy of local infections, consisting of injecting into a patient an appropriate amount of a compound of formula I, followed by local irradiation. According to this embodiment, R may be further defined as an amino acid or a peptide containing an OH or SH group, or a derivative thereof selected from the group consisting of esters and N-protected derivatives wherein the protecting group is carbobenzoxy, trityl and tert-butoxycarbonyl, and in which case Y is O or S when A is a covalent bond. Thus, conjugates of bacteriochlorophyll a with serine and immunoglobulin-G and of chlorophyll a with serine have been prepared and found to be active against bacteria.
The invention further relates to a method of sterilization of human blood for transfusion purposes, consisting of treating samples of human blood with an appropriate amount of a compound of formula I, followed by irradiation. According to this embodiment, R may be further defined as an amino acid or a peptide containing an OH or SH group, or a derivative thereof selected from the group consisting of esters and N-protected derivatives wherein the protecting group is carbobenzoxy, trityl and tert-butoxycarbonyl, and in which case Y is O or S when A is a covalent bond.